Rotavirus is the major cause of severe childhood gastroenteritis world wide. The medical significance of this virus has prompted extensive efforts to develop effective vaccines for the past twenty years. The ultimate goal of this project is to obtain detailed information about the molecular biology of rotavirus replication. Using an in vitro replication system that we have developed, we want to address the following fundamental questions: (i) What is the specific role(s) of each individual protein of the viral polymerase complex in virus transcription and genome replication? (ii) Which signal(s) directs the viral polymerase to replicate only the virus RNAs? (iii) How do viral proteins communicate among themselves during RNA replication? The answers to these questions will not only further our understanding of rotavirus replication at the molecular level, but should provide new guidelines for the development of a reverse genetic system for rotaviruses. During the past year, we have made significant progress towards the goals of this project. Using various mutated T7 synthetic rotavirus RNAs, we have demonstrated that the RNA secondary structure is crucial for RNA replication. Specifically, interactions between the 5'- and 3'- ends of the RNA are required for efficient replication. We also dissected rotavirus replication into two distinct steps, initiation and elongation. These new findings indicate that rotavirus RNA replication is more complicated than thought previously. Currently, we are investigating the identity of the interaction between 5'- and 3'- ends of template RNA and how it affects replication and specific proteins involved in initiation and elongation.